Lipofectamine 3000 Reagent
Generation and transfection of induced pluripotent
stem cells (iPSCs)
Induced pluripotent stem cells (iPSCs) hold immense promise for the
future of regenerative medicine and personalized therapeutic treatments
for a myriad of diseases and conditions. Invitrogen Lipofectamine 3000
Transfection Reagent was developed to unleash the power of stem cells by
providing a highly efficient, cost-effective nucleic acid delivery alternative
to electroporation. This advanced lipid nanoparticle technology minimizes
the stress on cells caused by electroporation, simplifies the reprogramming
workflow, and enables advanced gene editing technologies.
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™
Lipofectamine 3000 reagent is designed to provide:
• Superior efficiency—for the broadest spectrum of
difficult-to-transfect cells
• Low toxicity—gentle on cells for improved viability
• Versatility—single kit for DNA, RNA, and cotransfection
“Successfully transfected GT1-1 cells.
Never achieved with any other reagent
except viruses and nucleofectors.”
—Julien Sebag, PhD
Vanderbilt University
2
Thermo Fisher Scientific | Lipofectamine 3000 reagent
Contents
Generation of iPSCs from donor-derived fibroblasts
4
Genome editing of pluripotent stem cells 6
Transfection of pluripotent stem cells using
Lipofectamine 3000 reagent
7
Detailed protocol for reprogramming human fibroblasts
8
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3
Generation of iPSCs from donorderived fibroblasts
Reprogramming of fibroblasts
Patient-derived iPSCs offer exciting potential by enabling access to cell populations that are otherwise unavailable from living
donors. Efficient reprogramming of donor somatic cells to iPSCs plays a key role in the realization of this potential. In this
study, fibroblasts from skin biopsies of three donors were reprogrammed to iPSCs using the Invitrogen Epi5 Episomal iPSC
Reprogramming Kit* and Lipofectamine 3000 reagent (Figures 1 and 2). Reprograming efficiencies equivalent to those seen
with electroporation were observed (Figure 3). This advancement in delivery helps to minimize stress on the cells caused by
electroporation, simplifies the reprogramming workflow, and results in transgene-free, virus-free human iPSCs at efficiencies
in the range of 0.04% to 0.3%.
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CULTURE
ENGINEERING
DIFFERENTIATION
CHARACTERIZATION
Lipofectamine 3000
reagent and the Epi5
Episomal iPSC
Reprogramming Kit
enable highly efficient
reprogramming of
somatic cells without
the need for
electroporation.
CULTURE
ENGINEERING
DIFFERENTIATION
CHARACTERIZATION
Lipofectamine 3000
transfection reagent also
improves the cleavage
efficiency with CRISPR
vectors on pluripotent
stem cells, yielding a
2-fold increase in the
efficiency of genetic
modifications.
Indel in
genomic DNA
Adult cells
Lipofectamine 3000 reagent
can deliver DNA into ESCs
and iPSCs in culture.
™
CULTURE
ENGINEERING
DIFFERENTIATION
CHARACTERIZATION
Lipofectamine 3000 reagent
can deliver DNA into
difficult-to-transfect
differentiated cells in culture.
Ectoderm
iPSCs
Reprogramming
factors
Mesoderm
Endoderm
Figure 1. Lipofectamine 3000 reagent for pluripotent stem cell applications. High transfection efficiency enables Lipofectamine 3000 to be used for
a variety of applications, including reprogramming of fibroblasts, gene editing, and transfection of difficult cells.
* Designed by Dr. Okita in the laboratory of Professor Yamanaka at the Center for iPS Cell Research and Application (CiRA), Kyoto University.
4
Thermo Fisher Scientific | Lipofectamine 3000 reagent
• Day –1: Seed cells on
Geltrex matrix–coated
dishes
• Day 0: Transfect in Fibroblast Medium for 24 hours
• Day 15–20: Culture in E8
Medium (change daily)
• Day 1–14: Culture in N2B27 Medium with 100 ng/mL bFGF
(change daily)
• Days 21+: Manually pick
and expand colonies
Day –1
Day 0
Fibroblast Medium
Day 14
N2B27 Medium with bFGF (100 ng/mL)
Day 21
Essential 8 Medium
Geltrex matrix
Lipofectamine 3000
Somatic cell
Colony formation
begins
Identify ESC-like
colonies
Manually pick
and expand
+
Epi5 Episomal iPSC
Reprogramming Kit
Figure 2. Protocol outline for generating iPSCs using the Epi5 reprogramming kit and Lipofectamine 3000 reagent. Colonies can be picked and
expanded less than 21 days after fibroblast transfection.
A
B
Day 14
Day 17
BJ
HDFn
HDFa
Lipofectamine
3000 reagent
Neon system
electroporation
Figure 3. Reprogramming efficiency of Lipofectamine 3000 reagent compared to electroporation. BJ fibroblasts as well as neonatal (HDFn) and
adult (HDFa) human dermal fibroblasts were reprogrammed to iPSCs by transfection of Epi5 vectors using either Lipofectamine 3000 reagent or the
Invitrogen Neon Transfection System. Colonies were (A) visualized by brightfield microscopy and (B) stained for alkaline phosphatase.
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thermofisher.com/3000
5
Genome editing of pluripotent
stem cells
Genome editing technology derived from clustered regularly interspaced short palindromic repeats (CRISPRs) allows precise
cleavage of DNA at specific loci. However, the effectiveness of genome editing is contingent upon the intrinsic properties
of the locus of interest, efficiency of delivery, and the painstaking downstream processes of generating stable cell lines and
knockout models to study the phenotypic effects of the genetic modifications. Here we demonstrate that Lipofectamine
3000 reagent can deliver plasmid DNA into various iPSC clones for genome engineering purposes using targeted CRISPR
vectors. More than a 2-fold increase in gene modification efficiency compared to Invitrogen Lipofectamine 2000 reagent
was observed using Lipofectamine 3000 reagent, and with a lower amount of lipid (Figure 4).
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F1 origin
Pol III
term
pUC origin
OFP
AmpR
2A
Dosage (μL)
CRISPR Nuclease OFP
Reporter 9,219 bp
GFP control
vector
tracrRNA
CAAAA
Lipofectamine
3000 reagent
TK pA
CACCG
1 kb ladder
U6 promoter
Lipofectamine
2000 reagent
Design insert for GeneArt CRISPR vector
0.3
0.5
0.15
0.3
0.3
2.5
3.6
5.5
4.0
0
500 bp
400
PCMV
300
Cas9 (with NLS1 and NLS2)
200
Deliver with Lipofectamine 3000 reagent
100
Gene
modification
efficiency (%)
Measure cleavage efficiency using GeneArt
Genomic Cleavage Detection (GCD) Kit
™
Cas9
Indel in
genomic DNA
Target-specific crRNA
G
G C A U U UC G G U A UG C A U A UG A A U
C G T A A A G C C A T A C G T A T A C T A C C
G C A T T T C G G T A T G C A T A T G A N G G
Target genomic loci
Colony screening and expansion
6
Thermo Fisher Scientific | Lipofectamine 3000 reagent
PAM
Figure 4. Lipofectamine 3000 reagent
improves cleavage efficency of CRISPR
vectors in iPSCs. An Invitrogen GeneArt
CRISPR vector targeting a specific locus was
transiently transfected using Lipofectamine
2000 or Lipofectamine 3000 reagent. Cleavage
efficiency was assessed using the Invitrogen
GeneArt Genomic Cleavage Detection Kit.
G
U A
C G
G C
G C
U A
G C
A A
C G
G
A
U U U U G
A U
U A
U
C G
G
G C A
A
A
A
A
G UG
A
A U
A
U A
A
U A
G
U A
U
U A
U
tracrRNA
G U
C
A A A G G C U A G UC C G U U A UC A A
™
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Transfection of pluripotent stem cells
using Lipofectamine 3000 reagent
Lipofectamine 3000 Transfection Reagent was used to deliver DNA into difficult-to-transfect stem cells
(H9 ESCs and iPSCs) growing as colonies in Gibco Geltrex matrix–coated plates (Figure 5). Transfection efficiencies
of 40–70% were observed, with high mean fluorescence intensity (Figure 6).
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Day 0
Timeline
Rolling
direction
Seeding aggregates
to 12-well plates
2
$
Vortex 2–3 sec
Diluted DNA
3
Day 1
Treat with collagenase for
5 min; cut colonies into
evenly sized aggregates
with roller
Steps
Seed cells to be 70–90%
confluent at transfection
$
Diluted Lipofectamine 3000
Medium
Cells
Geltrex matrix–
coated 6 cm plate
1
Transfer medium
containing aggregates and
let settle by gravity
4
5
Incubate cells overnight;
change medium and then
perform transfection
$
$
Dilute Lipofectamine 3000
Reagent in Opti-MEM
Medium (2 tubes)–mix well
Prepare master mix of DNA
by diluting DNA in OptiMEM Medium, then add
P3000 Reagent–mix well
Add diluted DNA to
each tube of diluted
Lipofectamine 3000
Reagent (1:1 ratio)
Incubate
Procedure Details (Two Reaction Optimization)
Component
96-well
Adherent cells
1–4 × 104
Opti-MEM
5 μL × 2
25 μL ×
0.15 and 0.3μL
0.75 and 1.5
®
Medium
Lipofectamine
Opti-MEM
®
®
3000 Reagent
Medium
10 μL
0.2 μg
1 μg
P3000™ Reagent (2 μL/ μg DNA)
0.4 μL
2 μL
1. SSEA4 staining for pluripotency
Diluted DNA (with P3000
™
Reagent)
Day 2-4
5 μL
25 μL
5 μL
25 μL
96-well
24-well
2. Fluorescence imaging
Diluted Lipofectamine
®
3000 Reagent
3. Flow cytometry
Incubate for 5 minutes at room temperature.
$
$
Add DNA-lipid
complex to cells
DNA-lipid complex
10 μL
50 μL
DNA amount
100 ng
500 ng
P3000™ Reagent
0.2 μL
Lipofectamine
7
50 μL
DNA (0.5–5 μg/ μL)
Component (per well)
6
24-well
0.5–2 ×
$
Visualize/analyze
transfected cells
®
3000 Reagent used
0.15 and 0.3μL
1 μL
0.75 and 1.5
Incubate cells for 2–4 days at 37°C. Then, analyze transfected cells.
For support, visit www.lifetechnologies.com
Figure 5. Protocol outline for transfection of pluripotent stem cells with Lipofectamine 3000 reagent. Cells were transferred into multiwell plates
from Geltrex matrix–coated plates and transfected with Lipofectamine 3000 reagent. Transfection efficiency was assessed using flow cytometry and
fluorescence imaging.
A
B
DNA: 1.0 µg
Lipofectamine 3000: 1.5 µL
SSEA+/GFP+: 42%
GFP MFI 247344
DNA: 1.3 µg
Lipofectamine 3000: 1.5 µL
SSEA+/GFP+: 69%
GFP MFI 456741
Figure 6. Transfection of stem cells. (A) H9 ESCs or (B) iPSCs were transfected using Lipofectamine 3000 reagent. Cells were visualized by
fluorescence microscopy and processed using flow cytometry to determine transfection efficiency.
Summary
Lipofectamine 3000 Transfection Reagent was developed to improve transfection efficiency in difficult-to-transfect cells. In
this study, we demonstrated that using a superior transfection reagent such as Lipofectamine 3000 reagent in conjunction
with the Epi5 Episomal iPSC Reprogramming Kit enables highly efficient reprogramming of somatic cells without the need for
electroporation.
thermofisher.com/3000
7
Detailed protocol for reprogramming
human fibroblasts
Lipofectamine 3000 Transfection Reagent, used with the Epi5 Episomal iPSC Reprogramming Kit, enables highly efficient
reprogramming of somatic cells without electroporation.
Reprogramming timeline
8
Day –1
Day 0
Days 1–14
Days 15–20
Days 21+
Seed cells on Geltrex
matrix–coated 6-well
dishes
Transfect in fibroblast
growth medium for 24 hr
N2B27 Medium with
100 ng/mL FGF
(change medium daily)
Essential 8 Medium
(change medium daily)
Colonies ready for picking for
further culture and expansion
Cells and reagents
Cat. No.
Equipment
Epi5 Episomal iPSC Reprogramming Kit
A15960
Sterile cell culture hood (i.e., biosafety cabinet) equipped
with a stereomicroscope
Lipofectamine 3000 Transfection Reagent
L3000015
Inverted microscope
Opti-MEM I Reduced Serum Medium
31985062
Incubator set at 37°C, 5% CO2
Basic Fibroblast Growth Factor (bFGF), Recombinant
Human Protein
PHG0261
Water bath set at 37°C
KnockOut Serum Replacement
10828010
Sterile serological pipettes (5 mL, 10 mL)
Essential 8 Medium
A1517001
Centrifuge
DMEM/F-12, GlutaMAX Supplement
10565018
15 mL centrifuge tubes
N-2 Supplement (100X)
17502048
6-well tissue culture treated plates
B-27 Supplement (50X), Serum Free
17504044
10 µL, 200 µL, and 1,000 µL micropipettors with tips
100X MEM Non-Essential Amino Acids (NEAA)
11140050
β-Mercaptoethanol
21985023
Fetal Bovine Serum, embryonic stem cell–qualified
16141061
DMEM, High Glucose, GlutaMAX Supplement
10566016
0.05% Trypsin-EDTA (1X), phenol red
25300054
Geltrex LDEV-Free, hESC-Qualified, Reduced Growth
Factor Basement Membrane Matrix
A1413301
Dulbecco’s PBS (DPBS) without Calcium and Magnesium
14190
Thermo Fisher Scientific | Lipofectamine 3000 reagent
Preparation of cells and reagents
I. Media preparation
Fibroblast Medium
To prepare 500 mL of Fibroblast Medium, aseptically mix the
following components. Fibroblast Medium can be stored at
4˚C for up to 1 month.
DMEM
445 mL
Fetal bovine serum (FBS), ESC-qualified 100X MEM NEAA (10 mM)
50 mL
5 mL
N2B27 Medium
To prepare 500 mL of complete N2B27 Medium, aseptically
mix the following components. N2B27 Medium (without
bFGF) can be stored at 2–8°C for up to 1 week.
479 mL
DMEM/F-12
N-2 Supplement (100X)
5 mL
B-27 Supplement (50X)
10 mL
™
100X MEM NEAA (10 mM)
β-Mercaptoethanol (1,000X)
5 mL
908 μL
bFGF (100 µg/mL)
1. bFGF stock solution: To prepare 1 mL of
100 μg/mL bFGF solution, aseptically mix the following
components:
bFGF
100 µg
DPBS without Ca and Mg 2+
2+
KnockOut Serum Replacement
999 µL
1 µL
2. Aliquot and store the bFGF solution at –20°C for up
to 6 months.
3. A total of 1.0 µL of bFGF is needed for every mL of
N2B27 Medium to be used. Prepare the N2B27 Medium
without bFGF, and then supplement with fresh bFGF to
a final concentration of 100 ng/mL when the medium
is used.
Essential 8 Medium
1. To prepare 500 mL of Gibco Essential 8 Medium, thaw
the frozen Essential 8 Supplement at 2–8°C overnight.
Do not thaw the frozen supplement at 37°C.
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2. Mix the thawed supplement by gently inverting the vial
a couple of times, remove 10 mL from the bottle of
Essential 8 Basal Medium, and then aseptically transfer
the entire contents of the Essential 8 Supplement to the
bottle of Essential 8 Basal Medium.
3. Swirl the bottle to mix and to obtain 500 mL of
homogenous complete medium.
4. Complete Essential 8 Medium can be stored at 2–8°C
for up to 2 weeks. Before use, warm complete medium
required for that day at room temperature until the bottle
is no longer cool to the touch. Do not warm the medium
at 37°C.
II. Preparation of Geltrex matrix–coated dishes
1. Thaw Geltrex matrix at 4°C overnight.
2. Remove DMEM/F-12 from 4°C storage. Work
asceptically on ice to prevent polymerization. Dilute the
Geltrex matrix with an equal amount of DMEM/F-12 (1:1)
and mix gently. Aliquot into microcentrifuge tubes and
freeze immediately at –20°C.
3. Prepare a working stock solution on ice from the 1:1
diluted Geltrex matrix with additional DMEM/F-12. For
general growth, a final dilution of 1:100 is recommended.
Cover the whole surface of each well of a 6-well dish
with 1.5 mL of the working stock of Geltrex matrix.
4. Incubate the coated culture vessels for 1 hour at 37°C.
Transfer the plates or dishes to a laminar flow hood
and allow them to equilibrate to room temperature
(about 1 hour) before use. If the coated plates or dishes
are not used right away, seal with Parafilm wrap and
store at 4°C for up to 2 weeks. Care must be taken
to prevent these stored dishes from drying out or
being contaminated.
™
thermofisher.com/3000
9
III. Fibroblast culturing
1. Prewarm Fibroblast Medium in a 37°C water bath.
2. Wear eye protection, as cryogenic vials stored in liquid
nitrogen may explode when warmed.
3. Wear ultralow-temperature cryogenic gloves. Remove
the cryogenic vial of BJ fibroblasts from the liquid
nitrogen storage tank using metal forceps.
17. Aspirate the DPBS wash and add 2 mL of 0.05%
trypsin-EDTA, at room temperature, to the culture vessel
(adjust volume of trypsin up or down for appropriate
culture vessel size). Incubate the culture in a 37°C, 5%
CO2 incubator for approximately 3 to 5 minutes. Observe
the cells under a microscope and stop incubation when
the cells start to round up.
5. Immerse vials in a 37°C water bath. Swirl gently.
Note: Do not submerge the cap.
18.Add 4 mL prewarmed Fibroblast Medium to the culture
dish to stop trypsinization. Wash off all the cells from
the vessel surface by flushing the suspension several
times to harvest all the cells in a single cell suspension.
Transfer the cell suspension to a 15 mL centrifuge tube.
6. When only an ice crystal remains, remove the vial from
the water bath.
19.Centrifuge at 200 x g for 4 minutes and aspirate
the supernatant.
7. Spray the outside of the vial with 70% ethanol and place
in the hood.
20.Resuspend the cell pellet with 2 to 3 mL of fresh
prewarmed Fibroblast Medium and pipet up and down
to create a single cell suspension.
4. Roll the vial between your hands for about
10–15 seconds to remove frost.
8. Pipet cells into a 50 mL conical tube with a
1 mL micropipettor.
9. Using a 10 mL pipette, add 10 mL of prewarmed
Fibroblast Medium to the 50 mL conical tube dropwise
(to avoid osmotic shock to cells) while gently swirling the
conical tube. Pipette up and down gently to mix.
10.Centrifuge at 200 x g for 4 minutes and aspirate
the supernatant.
11. Resuspend the cell pellet in the appropriate amount of
Fibroblast Medium. Typically seed the contents of the
vial in 5–7 mL of medium in a T25 tissue culture treated
flask.
12.Incubate the BJ fibroblast cultures in a 37°C, 5% CO2
incubator overnight.
13.The following day, aspirate off spent medium and
replace with fresh, prewarmed Fibroblast Medium.
Change medium every other day until the flask is
approximately 70% confluent.
14.When 70% confluency is reached, the cultures must be
split and reseeded for creating cell banks, or seeded
directly for Epi5 kit transfection. It is recommended to
use a portion of early-passage cells for experimentation.
15.To split fibroblast cultures, aspirate the Fibroblast
Medium from culture vessel.
16.To wash, add 5 mL of room-temperature DPBS without
CaCl2 and MgCl2 (divalent cations must not be present
for proper dissociation of the fibroblasts from the culture
dish) and allow the wash solution to remain at room
temperature for 5 minutes.
10 Thermo Fisher Scientific | Lipofectamine 3000 reagent
21.Prepare a cell count and seed the appropriate amount
of cells into a new tissue culture treated dish with the
appropriate volume of medium. Typically a 1:6 split can
be used for regular maintenance every 3 to 4 days.
IV. Epi5 episomal reprogramming
Seeding human fibroblasts on Geltrex matrix–coated dishes
Plate 50,000 to 100,000 cells per well into a 6-well plate
at ~30–60% confluence (see Figure 7) in 2 mL Fibroblast
Medium and culture overnight at 37˚C and 5% CO2.
Transfection of human fibroblasts in a 6-well dish using
Lipofectamine 3000 reagent
1. Prewarm Gibco Opti-MEM I Reduced-Serum Medium
to room temperature and prepare Tube A and Tube B as
described below.
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2. Add 1.2 µL each of the two Epi5 Reprogramming
Vector mixes (2.4 µL total) to 118 µL Opti-MEM medium
in a 1.5 mL microcentrifuge tube labeled Tube A. Add
4.8 µL of P3000 Reagent and mix well.
™
™
3. Dilute 3.6 µL Lipofectamine 3000 reagent in
121 µL prewarmed Opti-MEM medium in a 1.5 mL
microcentrifuge tube labeled Tube B.
Note: Lipofectamine 3000 reagent diluted in
Opti-MEM medium should be used within 15 minutes
of dilution. Longer times can result in a loss of
transfection efficiency.
100,000 cells/6 well
HDFn
50,000 cells/6 well
Removal of transfection reagent and recovery of cells
1. 24 hours posttransfection, aspirate the medium from the
plates. Add 2 mL N2B27 Medium supplemented with
100 ng/mL bFGF (added fresh prior to use) to each well.
2. Change the N2B27 Medium every day for a total of 14
days by replacing the spent medium with 2 mL N2B27
Medium supplemented with 100 ng/mL bFGF per well.
HDFa
Transition to Essential 8 Medium
1. Aspirate the spent N2B27 Medium on Day 14 and
replace it with complete Essential 8 Medium. Resume
medium changes every day at 2 mL per well.
Figure 7. Seeding densities of neonatal (HDFn) and adult (HDFa)
human dermal fibroblasts.
Preparation of contents of Tube A and Tube B on a per-well
basis of a 6-well dish is outlined in the table below:
4. To prepare a transfection master mix, add the contents
of Tube A to Tube B and mix well.
2. Observe the plates every other day under a microscope
for the emergence of cell clumps, indicative of
transformed cells. Within 15 to 21 days posttransfection,
the iPSC colonies will grow to an appropriate size for
transfer.
3. Colonies are distinct by Day 21 and can be picked for
further culture and expansion. An example of results is
shown in Figure 8.
5. Incubate the transfection master mix for 5 minutes
at room temperature.
6. Mix one more time and add the entire 250 µL of
transfection master mix to each well of human fibroblasts
seeded on Geltrex matrix–coated plates containing 2 mL
fresh fibroblast growth medium.
50,000 cells
Volume
Tube A
Epi5 reprogramming vectors
1.2 μL
Epi5 p53 and EBNA vectors
1.2 μL
Opti-MEM medium
118 μL
P3000 reagent
4.8 μL
Tube B
Lipofectamine 3000 reagent
3.6 μL
Opti-MEM medium
121 μL
HDFa
Component
100,000 cells
HDFn
7. Incubate the plates in a 37°C, 5% CO2 incubator
for 24 hours.
Figure 8. Alkaline phosphatase–positive colonies obtained after
reprogramming of neonatal (HDFn) and adult (HDFa) human dermal
fibroblasts.
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For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. B-27 is a trademark
of Southern Illinois University. Essential 8 is a trademark of Cellular Dynamics International, Inc. Parafilm is a trademark of Bemis
Company, Inc. CO019607 1215